Electrochemical assay of xanthine oxidase



United States Patent ()fifice 3,356,555 Patented Jan. 30, 1968 3,366,555 ELECTROCHEMICAL ASSAY F XANTHINE OXIDASE David N. Kramer, Stevenson, and George G. Guilhanlt,

Edgewood, Md., and Paul L. Cannon, .lr., Harrisburg, Pa., assignors to the United States of America as represented by the Secretary of the Army No Drawing. Filed Mar. 1, 1965, Ser. No. 436,389 2 Claims. (Cl. 204-1) The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment to us of any royalty thereon.

This invention relates to a new method for the determination of the amount of organoarsenic compounds in an aqueous solution. With our new method one can obtain a quantitative analysis of solutions containing military agents such as dichloro (2-chlor0vinyl) arsine (Lewisite) and diphenylaminochloroarsine (Adamsite). By the use of our method, the concentration of the above military chemicals can be determined when present in an unknown solution in a concentration of 0.1 to 10 micrograms per milliliter.

Our method is similar to the method disclosed and claimed in S.N. 266,461 filed on Mar, 19, 1963 under the names Paul L. Cannon, Jr. and David N. Kramer, now U.S. Patent No. 3,275,534. In this prior method, it was disclosed that the inhibition of the enzyme, cholinesterase by oragnophosphorus compounds could be used as a method for determining the amount of organophosphorus compound present in the added unknown solution.

We have found that another enzyme, xanthine oxidase can be used in a similar manner to determine the amount of organoarsenic compounds present in an unknown solution.

It is well known that xanthine oxidase catalyzes the oxidation of hypoxanthine to uric acid and hydrogen peroxide. When using the apparatus described in S.N. 266,461 it is possible to establish a voltage-time curve for the reduction in the potential of the hypoxanthine-xanthine oxidase system similar to FIGURE 2 of the above co-pending case. When no organoarsenic compound is added to the system, the reduction in the potential across the cell will be relatively large per unit of time. When an organoarsenic compound is added to the system the xanthine oxidase is inhibited in its activity to a greater or lesser degree in a manner which is proportional to the amount of the organoarsenic compound which has been added. Thus, it is possible to prepare a calibration chart for this system analogous to FIGURE 3 of the above case and the unknown concentration of the organoarsenic compound can be determined,

In using our method, the temperature (normally 25 C.) was controlled to within plus and minus 1 C. and the method must be carried out in the absence of cyanide ion which interferes with the determination.

Example 1 Twenty-five milliliters of a 1 10 M hypoxanthine solution in a buffered solution at pH 6.76 (prepared by mixing disodium hydrogen phosphate, citric acid and potassium chloride in distilled water as used by Elving et al., I. Am. Chem. Soc. 79:2697 (1957)) was placed in a 50 ml. beaker along with 1.0 ml. of the organoarsenic solution to be determined and the apparatus shown in FIG. 1 of the above co'pending case was applied to this solution. A constant current of 3.8 microamperes was applied across the electrodes and the potential of the plati num anode versus the reference electrode was automatically recorded. At zero time, 1.0 ml. of a 0.1 unit per ml. solution of xanthine oxidase is added to effect the oxidation of the substrate By resorting to the predetermined calibration plots of voltage change per time change versus concentration of inhibitor, one can obtain the concentration originally present in the solution to be determined.

Our method is not limited in its use to the above mentioned organoarsenic compounds but is equally applicable to diphenylchlorarsine, methyldichloroarsine ethyldichloroarsine and phenyldichloroarsine as well as any other organoarsenic compound which exhibits the property of inactivating xanthine oxidase.

The pH of the buffered solution must be approximately neutral. A range of pH from 68 has been found to be acceptable with the preferred pH being about 6.76.

We claim:

1. A method for the detection of organoarsenic compounds which comprises:

(a) applying a low direct current across a negative and a positive electrode immersed in a stirred buffered aqueous solution of one of said compounds and hypoxanthine at a pH range of 6-8 having a reference electrode therein,

(b) recording the potential between said positive electrode and said reference electrode,

(c) adding xanthine oxidase to said solution,

((1) recording the ratio of the change in potential between said positive electrode and said reference electrode to the change in time from the addition of said xanthine oxidase, said ratio being inversely proportional to the concentration of said compound in said buttered solution.

2. A method for the detection of a compound selected from the group consisting of dichloro (2-chlorovinyl) arsine, diphenylaminochloroarsine, diphenylchloroarsine, methyldichloroarsine, ethyldichloroarsine, and phenyldichloroarsine which comprises:

(a) applying a low direct current across a negative and a positive electrode immersed in a stirred buffered aqueous solution of one of said compounds and hypoxanthine at a pH of about 6.76 having a reference electrode therein,

(b) recording the potential between said positive electrode and said reference electrode,

(c) adding xanthine oxidase to said solution,

(d) recording the ratio of the change in potential between said positive electrode and said reference electrode to the chan e in time from the addition of said xanthine oxidase, said ratio being inversely proportioned to the concentration of said compound in said bufi'ered solution.

References Cited UNITED STATES PATENTS 2,757,132 7/1956 Northrop 204- 3,049,411 8/1962 Gelman et a1 23232 I-IOlVARD S. WILLIAMS, Primary Examiner. T. TUNG, Assistant Examiner. 

1. A METHOD FOR THE DETECTION OF ORGANOARSENIC COMPOUNDS WHICH COMPRISES: (A) APPLYING A LOW DIRECT CURRENT ACROSS A NEGATIVE AND A POSITIVE ELECTRODE IMMERSED IN A STIRRED BUFFERED AQUEOUS SOLUTION OF ONE OF SAID COMPOUNDS AND HYPOXANTHINE AT A PH RANGE OF 6-8 HAVING A REFERENCE ELECTRODE THEREIN, (B) RECORDING THE POTENTIAL BETWEEN SAID POSITIVE ELECTRODE AND SAID REFERENCE ELECTRODE, (C) ADDING XANTHINE OXIDASE TO SAID SOLUTION, (D) RECORDING THE RATIO OF THE CHANGE IN POTENTIAL BETWEEN SAID POSITIVE ELECTRODE AND SAID REFERENCE ELECTRODE TO THE CHANGE IN TIME FROM THE ADDITION OF SAID XANTHINE OXIDASE, SAID RATIO BEING INVERSELY PROPORTIONAL TO THE CONCENTRATION OF SAID COMPOUND IN SAID BUFFERED SOLUTION. 